The present invention relates to a device and method of detecting microorganisms in a rapid manner and increasing the number of microorganisms detected during the culturing of a sample by the addition of resinous and non-resin adsorbents and molecular sieves in the growth media. These agents have been found to neutralize or remove inhibitory and antimicrobial substances found in samples and media. The invention also relates to specific non-resinous adsorbents and the methods of manufacturing them.
It is standard practice to detect the presence of microorganisms in samples by culturing samples in a liquid growth medium. Medical test samples include body fluids such as blood, urine and cerebral spinal fluid and other solid or semi-solid samples such as tissue fluidized in saline or other liquid reagents. Industrial samples include pharmaceuticals, foods or any product that must be tested for the presence or levels of microorganisms.
The detection of these microorganisms can be impaired by the condition of the samples themselves. For example, medical samples may contain levels of antibiotics due to treatment regimens, while it is known that serum, plasma and lysed erythrocytes naturally contain microbial inhibitors. Industrial samples such as pharmaceuticals and foods may also contain antimicrobials or preservatives that inhibit the growth of microorganisms. Additionally, when culture media is prepared, autoclaving of the media at very high temperatures under pressure can result in the formation of by-products toxic to microorganisms. Removal or neutralization of these inhibitory or bactericidal substances is necessary to detect microbial contamination.
The use of synthetic anionic exchange and nonionic adsorbent resins for removal of antimicrobial substances is well known and has been previously described for use in medical diagnostic procedures. In particular, these resins have been shown useful in the removal of antibiotics and other antimicrobials from blood samples. The removal of these inhibitors in medical samples allows for recovery and faster detection of microorganisms so that microbial identification and accurate antibiotic susceptibility testing can be performed.
Melnick et. al., U.S. Pat. No. 4,145,304, describes the use of synthetic anionic exchange and nonionic resins to remove antimicrobials, including antibiotics, from body fluids, thus allowing for recovery of pathogens using standard culture techniques. The resins described are coated with a nonionic detergent in order to selectively remove charged antibiotics while inhibiting adherence of bacteria to the resins. After treatment of the sample with the resin, the eluate is cultured in a growth media. The degree of binding of antibiotics by the resins is indicated to be dependent on the total exchange capacity, pore diameter, and surface area of the resin.
Waters, U.S. Pat. No. 4,632,902, herein incorporated by reference, describes an improvement over Melnick by incorporating ion exchange resins and non-functional adsorbent resins directly into the growth medium. Inhibitors removed include antibiotics administered to patients and naturally occurring inhibitors contained in serum, plasma, and lysed erythrocytes. The resins are not coated with a nonionic detergent or surfactant before use and the pore size of the resin is not critical.
The use of resins as described above indicates that detergent treatment of the resins, in particular, the nonionic polystyrene resin which is extremely hydrophobic, may or may not be necessary. The confusion may be due to the fact that nonionic polystyrene resins are generally pretreated with surfactants by the manufacturer prior to shipment in order to be used in hydrophilic environments. Therefore, treatment with surfactants or detergents is an absolute requirement to wet these resins, thus allowing for hydrophilic interaction, particularly with broth culture medium. If the hydrophobic resin is not treated, the resin will float at the top of a liquid culture medium without any interaction possible to bind antimicrobial components.
However, using adsorbents in culture media can cause nonspecific binding of proteins, carbohydrates and other media components to the adsorbent. If enough of these components are bound and therefore not accessible to the microorganisms, no microorganisms will be detected. This binding is dependent on the ratio of the adsorbent to media volume and concentration.
The removal of toxic by-products as well as growth components from medium influences its color. Depending on the amount of adsorbent material and the composition of the particular medium, it may become colorless, which may indicate an inability to support growth. However, the ability of the medium to support growth is not always indicated by color, as chemically defined or semi-defined media are colorless and can still maintain optimal growth performance.
The culture medium formulation is critical in providing optimized growth for a wide variety of microorganisms. This is particularly important when the production of carbon dioxide is used as the measure of microbial growth, for example, as described in U.S. Pat. No. 4,945,060. The proper ratio of growth promoting components is essential to provide optimal carbon dioxide production from a variety of microorganisms with diverse metabolic pathways in order to rapidly detect microbial growth. The removal of medium components by any adsorbent material must be carefully controlled to maintain the optimal carbon dioxide production required for detection of microorganisms, especially if no additional growth supplements, such as blood, are added.
The use of synthetic resins in culture medium improves recovery of microorganisms from body fluids, and especially from blood. These fluids are nutritious and therefore compensate for a lack of nutrients in the adsorbent containing culture medium. However, in situations where the test sample can add no nutritional value to the medium containing the resins, the ability to recover and detect microorganisms can be greatly compromised.
When antimicrobials are removed from or neutralized in culture medium, the degree of neutralization is dependent on a number of factors. These factors include the dilution ratio of the sample to the medium, the concentration of the antimicrobial in the sample, and the antimicrobial sensitivity of the particular organism contained in the sample. In the case of blood samples from patients on antibiotics, there is a maximum therapeutic level of a particular antibiotic that can be achieved that differs with each antibiotic. Therefore, in an optimal testing situation, the amount of adsorbent should vary with each sample.
Although synthetic resins are known to remove inhibitory substances in cultures containing body fluids, it is desirable to find other substances that perform a similar function on both body fluids and non-body fluid samples, such as foods and industrial products. The present invention removes, inhibits, or isolates antimicrobials in test samples, while retaining the components of the medium necessary to recover and detect microorganisms in a rapid manner.